title: Soluble and catalytically active endothelin converting enzyme-1 is present in cerebrospinal fluid of subarachnoid hemorrhage patients.
abstract: Endothelin converting Enzyme-1 (ECE-1) is essential for the production of Endothelin-1 (ET-1), which is associated with vasospasm following subarachnoid hemorrhage (SAH). We have previously demonstrated the presence of a catalytically active soluble form of ECE-1 in the media of endothelial cells. We aimed to determine if this form of ECE-1 exists in vivo, in cerebrospinal fluid (CSF) of SAH patients. We examined CSF taken from SAH subjects for the presence of soluble ECE-1 using a bradykinin based quenched fluorescent substrate assay. We obtained further confirmation by characterizing the CSF mediated cleavage products of BigET-1 and BigET₁₈₋₃₄ (6 μg/ml) using mass spectrometry. The specificity of cleavage was confirmed using the ECE-1 inhibitor CGS35066 5 nmol/L. SAH CSF samples had mean ECE-1 activity of 0.127 ± 0.037 μmols of substrate cleaved/μl of CSF/24 h. The C-terminal peptides generated upon the cleavage of BigET-1 and BigET₁₈₋₃₄ were detected 48 h after incubation of these substrates with CSF. Cleavage of these substrates was inhibited by CGS35066. Results of Western blots also produced strong evidence for the presence of truncated soluble ECE-1 in CSF. These results strongly suggest the presence of a truncated but catalytically active form of ECE-1 in the CSF of SAH subjects. Further studies are necessary to determine the biological significance of soluble ECE-1 in CSF of SAH subjects, including an association with vasospasm after SAH.
authors.affiliation: Department of Biochemistry & Molecular Biology, Building 77, Monash University, Wellington Rd, Victoria 3800, Australia;
authors.forename: Sanjaya
authors.initials: S
authors.lastname: Kuruppu
authors.forename: Sherry H-Y
authors.initials: SH
authors.lastname: Chou
authors.forename: Steven K
authors.initials: SK
authors.lastname: Feske
authors.forename: Sarah
authors.initials: S
authors.lastname: Suh
authors.forename: Iresha
authors.initials: I
authors.lastname: Hanchapola
authors.forename: Eng H
authors.initials: EH
authors.lastname: Lo
authors.forename: Mingming
authors.initials: M
authors.lastname: Ning
authors.forename: A Ian
authors.initials: AI
authors.lastname: Smith
mesh_terms: D016282:Aspartic Acid Endopeptidases / Q000134:cerebrospinal fluid*; D001572:Benzofurans / Q000494:pharmacology; D001920:Bradykinin / Q000378:metabolism; D019332:Endothelin-1 / Q000032:analysis* / Q000378:metabolism; D000072396:Endothelin-Converting Enzymes; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006849:Hydrocephalus / Q000134:cerebrospinal fluid; D013058:Mass Spectrometry; D008666:Metalloendopeptidases / Q000134:cerebrospinal fluid*; D063065:Organophosphonates / Q000494:pharmacology; D013345:Subarachnoid Hemorrhage / Q000134:cerebrospinal fluid* / Q000201:enzymology / Q000473:pathology
chemical_list: D001572:Benzofurans; C404932:CGS 35066; D019332:Endothelin-1; D004791:Enzyme Inhibitors; D063065:Organophosphonates; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes; D001920:Bradykinin